Identification of ZFTA as a Novel KLHL20 Substrate and Mechanistic Insights Into Fuzzy Binding of Disordered Peptides via Biosensor Analysis and Computational Modelling

Interactions between peptides based on a region in the zinc finger translocation associated (ZFTA) protein and the Kelch domain of Kelch-like protein 20 (KLHL20^Kelch) have been characterised by biosensor analysis, supported by AlphaFold2-based structure predictions of peptides bound to the protein. Residues critical for the interaction were identified. The analysis showed that all peptides exhibited relatively weak and complex interactions with KLHL20^Kelch. The original ZFTA peptide had a much higher affinity for KLHL20^Kelch than for the Kelch domain of KLHL12 (KLHL12^Kelch), indicating a specificity for KLHL20^Kelch. The estimated K_D ^app of 35 µM was like that for a 21-mer peptide derived from death-associated protein kinase 1, a known KLHL20 substrate. Removal of flexible C-terminal residues generated a 12-mer, predicted to form a stable helix. This reduced the affinity 100-fold. Removal of N-terminal residues resulted in a 10-mer predicted to be flexible, which had a similar affinity as the original 16-mer. The similar affinities for peptides representing different regions of ZFTA suggest that the recognition is feature specific rather than sequence specific. The interaction mechanism reflects "fuzzy binding", consistent with the role of KLHL20 as an adaptor protein in the ubiquitination of disordered protein substrates by Cullin-3 E3 ubiquitin ligase.